The invention comprises a process for improving the NADH-specificity of usually NADPH-dependent dehydrogenases which is useable particularly for obtaining dehydrogenasesxe2x80x94specifically short-chain dehydrogenases and preferably alcohol-dehydrogenasesxe2x80x94with an NADH-dependency corresponding to a kcat/KM-value for NAD+xe2x89xa720 suitable for preparative purposes. It includes dehydrogenases obtained thereby and the use thereof.
Dehydrogenases, particularly alcohol-dehydrogenases, (below abbreviated as ADHn) are valuable catalysts for obtaining chiral products by stereoselective reduction of prochiral ketones to the corresponding chiral alcohols. Commercially available are the corresponding enzymes from yeast (NAD-dependent), horse liver (AND-dependent) and Thermoanaerobium brockii (NADP-dependent) and also, for special substrates, for example steroid-dehydrogenases (NAD and NADP-dependent). Due to the quite limited substrate spectrum specifiy of these, additional new ADH""s have become commercially available in the last years which are especially well suited for preparative uses, for example, an (S)-specific ADH from Rhodococcus erythropolis (NAD-dependent) or (R)-specific enzymes from the species Lactobacillus. Both types of enzymes catalize a variety of ketone conversions with a high enantioselectivity. The enzymes from L. kefir (DE 40 14 573) or L. brevis are of particular interest since they are unique in leading to (R)-alcohols. However, there is a disadvantage in the use of these enzymes in that they require the co-enzyme NADP+ or respectively, NADPH since this co-enzyme is substantially less stable and more expensive (by a factor 5-10) than NAD+, or respectively, NADH.
It is the object of the present invention to provide a method for improving the NAD+ dependency of such ADHs, and which, moreover, is useable for improving dehydrogenases in general.
The initially mentioned inventive method which was developed for this purpose is characterized in that the bascity of the enzyme in the coenzyme-docking area is reduced by a suitable change in amino acid sequence using genetic engineering means. This reduction in basicity of the amino acid residues in the co-enzyme-docking area can be achieved particularly by an exchange of positively charged amino acid(s) for uncharged amino acid(s).
Alternatively, or additionally, the basicity of amino acids in the coenzyme docking area can be reduced by displacement of neutral or positively charged amino acid(s) with negatively charged amino acids. Of course, to achieve this reduction in basicity various combinations of these displacements are possible.
Further specialities of the invention are apparent from the patent claim and the following description.
The method is based on the fact that an acceptance of the coenzyme at the coenzyme-binding location is important for the participation of the coenzyme in the enzymatic redox-reaction and that the acceptance of NAD+ as compared to NADP+ can be improved by a reduction in basicity in the docking area of the enzyme through corresponding alterations of the amino acid sequence. Such alteration was done and examined by molecular engineering devices and a substantial improvement was achieved in this way as it will become apparent from the following description.
As a realization example, ADH from the Lactobacillus brevis (DSM 20 054) was used, wherein a coenzyme binding location in the N-terminus is assumed and whose sequence of the first 50 amino-acids in the N-terminal area is:
S-N-R-L-D-G-K-V-A-I10-I-T-G-G-T-L-G-I-G-L20-A-I-A-T-K-F-V-E-E-G30-A-K-V-M-I-T-G-R-H-S40-D-V-G-E-K-A-A-K-S-V50. (SEQ ID NO 21).
The complete sequence of the ADH""s subunit, which consists of four identical subunits, is published in the dissertation B. Riebel (1997, University of Dxc3xcsseldorf), and also the corresponding DNA sequence which served as a template for the mutations.
In this N-terminal sequence certain basicity-relevant amino acids were exchanged by established genetic engineering devices and the changes in the acceptance of NAD+ were determined basically with two values, the KM value and the kcat value. The KM value (Michaelis-Menten-constant [Mol/L]) can be considered to be a measure for the affinity of the co-enzyme to the enzyme. Preferably, the KM value is as small as possible. The kcat value (Mol formed product/mol enzymexc3x97sec) is a measure for the conversion, it should be as high as possible. A combined value, which takes both values into consideration is the quotient kcat/KM. This should be preferablyxe2x89xa720 for obtaining an enzyme suitable for preparative purposes.
In the following examples 1-4 basic amino acids were replaced by neutral amino acids at the locations R38, H39, K45 and/or K48. Characterization of the mutants reveals that it is actually possible to change the ADH""s coenzyme specificity (L. brevis) from NADP+ towards NAD+. Although only a limited number of amino acid exchanges were performed, the results show that the desired improvement of the NAD+ dependency by basicity reduction can be achieved.
An exchange of A9G (Alanine for Glycine; both are uncharged) additionally performed in the following examples was not done in order to change the basicity but out of additional stability considerations. It can be clearly shown that the NAD+ specificity changes, which were achieved by the exchange according to the invention for reducing the basicity, are not noticeably affected by the xe2x80x9caccompanying exchangexe2x80x9d (A96) as provided for in following examples.
Besides the inexaustive amino-acid exchanges performed close to the N-terminus area of the ADH from L. brevis, such an exchange can, of course, also be performed in the remaining amino acid chain (according to the above mentioned dissertation) and can be examined for a positive result with respect to the specificity for NAD+.
In general, the method according to the invention of altering dehydrogenases requires the knowledgexe2x80x94or methods that lead to the determinationxe2x80x94of the desired amino acid sequence which needs to be altered to improve a particular enzyme in order to permit the desired exchange of basic amino acids by uncharged or negatively charged amino acids or, respectively, an exchange of uncharged amino acids by negatively charged amino acids. The desired exchange area, which is useful for the improvement of the NAD-specificity, can be determined even with-out the previous knowledge of the coenzyme binding locationxe2x80x94by way of trial-and-error. Site-specific mutagenesis has become far easier with the now commercially available ready-to-use kits for performing essential steps in genetic engineering procedures. In the case of basic amino acids, particularly lysine and argenine can be exchanged, preferably for uncharged amino acids, such as glycine, alanine, valine, leucine, isoleucine, methionine, serine, tyrosine, or phenylalanine. In the case of negatively charged amino acids, particularly glutamate acids and aspartic acids may be considered.
The successive changes of the mutated enzymes were proven by the determination of the kinetic parameters for the co-enzyme NAD+, NAPD+, NADH and NADPH by means of the corresponding kinetic parameters for the ketone substrate (acetophenone) or, respectively, by the oxidation reaction for the alcohol (phenylethanol). Furthermore, maintenance of the enantioselectivety was examined using the example of reduction of acetophenon. In addition, temperature-optima and stability, pH optima and stability and the iso-electric point were determined and compared with the corresponding data for the non-mutated wild-type enzyme.
The wild type enzyme has the following properties:
A) Kinetic Data for the Coenzyme NADP+, NAD+, NADPH and NADH (Table 1):
Table 1 shows that the coenzyme NADH of the wild type of the ADH from L. brevis is not converted to an extent that it can e detected. Only very high concentrations of NAD+ with a slight activity are accepted so that the selectivity (kcat/KM) of the wild type for NAD+ (=7) in comparison with the value for NADP+ (=270) is at about 2.5%.
B) Temperature-optimum and -stability
The temperature optimum of the wild type-ADH is around 55xc2x0 C.; after a 24 hour incubation period at various temperatures, it still exhibits 100% residual activity at 30xc2x0 C.; at 37xc2x0 C. the residual activity is 50%.
C) pH Optimum and Stability
The pH optimum for the reduction of acetophenon with NADPH is 6.5. The optimum range is very narrow. Already at pH 6.0 or 7.0, there is only a residual activity of about 60%. For the oxidation of phenylethanol with NADP+ the optimum is at about 8.0 with a wider optimum range of 7-9. The wild-type enzyme exhibits highest stability when stored at pH 7.0 to 8.5.
D) Determination of the Iso-electric Point.
The iso-electric point of the wild type enzyme is at 4.95.
E) Batchwise Reduction of Acetophenon Under Coenzyme Regeneration and Determination of the Enantioselectivity of This Conversion.
The reduction of acetophenon with NADPH using the wild type enzyme with coenzyme regeneration shows after gaschromatographic separation exclusively the (R) isomere of phenylethanol.